Massively parallel and multiplex blood group genotyping using next-generation-sequencing.

OBJECTIVES: Thirty-six blood group systems are listed by the International Society of Blood Transfusion, containing almost 350 antigens. Most of these result from a single nucleotide polymorphism (SNP). Serology is the standard method for blood group typing. However, this technique has some limitations and cannot respond to the growing demand of blood product typing for a large number of antigens. Here we describe a blood group genotyping assay directly from whole blood samples using Next-Generation Sequencing (NGS), allowing the simultaneous identification of 15 SNPs associated with the blood group systems of 95 patients in a single run. DESIGN AND METHOD: After an automated DNA extraction, targets are amplified by multiplex polymerase chain reaction (PCRm). Two panels addressing 9 groups have been developed (MNS, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, and Colton), one for 8 SNPs, the other for 7 SNPs. For each sample, both panels corresponding to 14 amplicons (1 amplicon containing 2 SNPs) are pooled. Then a dual-indexed library is generated from each pool by linking Illumina adaptors directly onto amplicons, followed by sequencing using the MiSeq platform (Illumina). RESULTS: In a single experiment, 95 blood donor samples have been sequenced for the genes of interest. Among the 1425 targeted single nucleotide polymorphisms, 1420 were identified by sequencing, reflecting a coverage of 99.65%. The obtained data shows a good correlation (99% for all SNPs) with other blood group typing methods. Depending on the allele pairs analyzed, correlations vary between 97.12 and 100%. CONCLUSION: Next-Generation sequencing would supplement serological and molecular techniques and, in the near future, could replace it with complete and fast results acquisition for pre-screening and identification of rare blood bags.

Development and Validation of a Fully Automated Platform for Extended Blood Group Genotyping.

Thirty-five blood group systems, containing >300 antigens, are listed by the International Society of Blood Transfusion. Most of these antigens result from a single nucleotide polymorphism. Blood group typing is conventionally performed by serology. However, this technique has some limitations and cannot respond to the growing demand of blood products typed for a large number of antigens. The knowledge of the molecular basis of these red blood cell systems allowed the implementation of molecular biology methods in immunohematology laboratories. Here, we describe a blood group genotyping assay based on the use of TKL immobilization support and microarray-based HIFI technology that takes approximately 4 hours and 30 minutes from whole-blood samples to results analysis. Targets amplified by multiplex PCR were hybridized on the chip, and a revelation step allowed the simultaneous identification of up to 24 blood group antigens, leading to the determination of extended genotypes. Two panels of multiplex PCR were developed: Panel 1 (KEL1/2, KEL3/4; JK1/2; FY1/2; MNS1/2, MNS3/4, FY*Fy et FY*X) and Panel 2 (YT1/2; CO1/2; DO1/2, HY+, Jo(a+); LU1/2; DI1/2). We present the results of the evaluation of our platform on a panel of 583 and 190 blood donor samples for Panel 1 and 2, respectively. Good correlations (99% to 100%) with reference were obtained.

Polymer adhesive surface as flexible generic platform for multiplexed assays biochip production.

The present report describes the integration and application possibilities of a new microarray concept based on adhesive surface. The method was shown to enable the straightforward production of 384 and 1536-well plates modified with 100 and 25 spots per well, respectively. Such in-well densities were only possible thanks to the fabrication process which implies first the deposition of the microarray on a flat adhesive surface and then its assembly with bottomless 384 or 1536-well plates. The concept was also confronted to various applications such as oligonucleotide detection, localised cell culture onto spotted adhesion proteins and immobilisation of peptide or active antibodies for immunoassays. In the particular case of immunotesting, the study focused on liver diseases diagnosis and more particularly on the detection of either one liver cancer marker, the alpha-fetoprotein, or the detection of Hepatitis C Virus infection. In every cases, interesting performances were obtained directly in crude patient serum, proof of the robust and generic aspect of the platform.

Recent advances in multiplex immunoassays.

The present review reports on the lastest developments in multiplex immunoassays. The selected examples are classified through their detection strategy (fluorescence, chemiluminescence, colorimetry or labeless) and their assay format (standard microtiter plate, polymeric membranes and glass slides). Finally, the degree of integration in a complete system, incorporating fluid handling and detection was also taken into account.

Impact of immobilization support on colorimetric microarrays performances.

We report here a comparison of support materials for colorimetric hybridization assays on microarrays. Four surfaces with various chemistries and architectures (roughness and porosity) were evaluated: (i) bare and (ii) activated polystyrene surfaces classically used for ELISA; (iii) a double-sided adhesive support; and (iv) a porous nitrocellulose/cellulose acetate membrane. Each substrate was functionalized with a microarray of probes and subjected to an enzymatic colorimetric DNA hybridization test. Tests were carried out in a 96-well assembly suitable for automated high-throughput analysis. Colorimetry results, microscopy observations and a chemiluminescence study showed that the test efficiency not only depends on the surface probe density but that the capacity of the material to retain the colored enzymatic product is also a critical parameter. All parameters being considered, the adhesive coated surface proposes the best surface properties for efficient colorimetric microarrays.

Adhesive microarrays for multipurpose diagnostic tools.

We are reporting here a new technology for the straightforward production of integrated microarrays. The approach is based on the use of adhesive supports enabling (i) the immobilization of biomolecules as microarrays (up to 2500 spots per cm(2)) and (ii) the easy assembly of these microarrays with complex 3D structures such as 96-well bottomless microplates or polymer and glass microfluidic networks. The analytical performances of the system were demonstrated for sandwich protein detection (C-reactive protein) and hybridization assays, both in classical 96-well microplate format and microfluidic environment.

Electrochemical surface nanopatterning using microspheres and aryldiazonium.

A multistep procedure to prepare heterogeneous structured surfaces with contrasted chemical functionalities at the nanometer scale is presented. Aryldiazonium cations are used for the nanopatterning of electrodes to create hybrid surfaces. The nanopatterning procedure involves the auto-organization of a polystyrene (PS) beads layer at gold or glassy carbon electrode surfaces. The deposited beads layer permits masking of a fraction of the surface from a first aryldiazonium electrografting process. By subsequent removal of the PS beads, the ungrafted surface areas become available for either another aryl diazonium electrografting or a metal electrodeposition, leading to hybrid nanostructured surfaces.

Protein-diazonium adduct direct electrografting onto SPRi-biochip.

A direct protein immobilization method for surface plasmon resonance imaging (SPRi) gold chip arraying is exposed. The biomolecule electroaddressing strategy, previously demonstrated by our team on carbon surfaces, is here valuably involved and adapted to create a straightforward and efficient protein immobilization process onto SPRi-biochips. The proteins, modified with an aryl-diazonium adduct, are addressed to the SPRi chip surface through the electroreduction of the aryl-diazonium. The biomolecule deposition was followed through SPRi live measurements during the electrografting process. A specially designed setup enabled us to directly observe the mass increasing at the sensor surface while the proteins were electrografted. A pin electrospotting method, allowing the achievement of distinct sensing layers on gold SPRi-biochips, was used to generate microarray biochips. The integrity of the immobilized proteins and the specificity of the detection, based on antigen/antibody interactions, were demonstrated for the detection of specific antibodies and ovalbumin. The SPRi detection limit of ovalbumin using the electroaddressing of anti-ovalbumin IgG was compared with two other immobilization procedures, cystamine-glutaraldehyde self-assembled monolayer and pyrrole, and was found to be a decade lower than these ones (100 ng/mL, i.e., 2 nM).

Biochips: non-conventional strategies for biosensing elements immobilization.

The present article draws a general picture of non-conventional methods for biomolecules immobilization. The technologies presented are based either on original solid supports or on innovative immobilization processes. Polydimethylsiloxane elastomer will be presented as a popular immobilization support within the biochip developer community. Electro-addressing of biomolecules at the surface of conducting biochips will appear to be an interesting alternative to immobilization processes based on surface functionalization. Finally, bead-assisted biomolecules immobilization will be presented as an open field of research for biochip developments.

On-chip chemiluminescent signal enhancement using nanostructured gold-modified carbon microarrays.

An original method for the enhancement of chemiluminescent (CL) on-chip detection of protein and oligonucleotides is presented. This enhancement is based on the electrodeposition of a gold nanostructured layer onto a screen-printed (SP) carbon microarray prior to the immobilization of biomolecules through a well-established diazonium adduct electrodeposition. Morphological studies of the Au layer (optical and atomic force microscopy) show that the metal film is composed of nanostructured 800 nm diameter particles covering the entire graphite surface and yielding a high surface area. Using these modified SP microarrays, enhancement factors of 229 and 126 were obtained for prostate-specific antigen (PSA) and p53 oligonucleotide detection, respectively. These enhancements were associated with three different phenomena: an enhancement of the catalyzed chemiluminescent reaction by the gold surface, an increase of the specific surface area for immobilization of the probe biomolecules, and an opposite quenching effect due to the overlapping of the gold absorption and CL emission peaks. For free PSA and target oligonucleotide detection, enhanced performances were obtained, giving detection limits of 5 ng/mL and 0.1 nM, respectively.

Direct electrochemical addressing of immunoglobulins: immuno-chip on screen-printed microarray.

An original immobilisation technology is presented for the development of chemiluminescent protein biochips, suitable for measurement in complex matrices. The immobilisation strategy involved is based on diazotated aniline derivatives, which could be electro-addressed, thus creating a covalent linkage with a conducting material surface. The present electrochemical system is a cost effective and mass-produced carbon paste screen-printed (SP) microarray composed of eight 0.2 mm2 working electrodes, one carbon pseudo-reference electrode and one auxiliary electrode. Rabbit immunoglobulins (IgG) were chemically modified with an aniline derivative (4-carboxymethylaniline) in order to be easily electro-grafted to the SP microarray surface. The possibility of successively electro-address the eight sensing layers of a particular array, with a good reproducibility (more than 80%) and without loss of reactivity was demonstrated. Moreover, these immobilised proteins were subsequently used as a capture agent for the determination of rheumatoid factor (RF) in human sera. The absence of non-specific signal or interference problem enabled the detection of RF values in complex samples in the 5.3-485 IU/ml range with a good correlation with the standard Auraflex ELISA test method.

Diazonium-protein adducts for graphite electrode microarrays modification: direct and addressed electrochemical immobilization.

Diazonium cation electrodeposition was investigated for the direct and electro-addressed immobilization of proteins. For the first time, this reaction was triggered directly onto diazonium-modified proteins. Screen-printed (SP) graphite electrode microarrays were studied as active support for this immobilization. A 10-microelectrode (eight working electrodes, 0.2 mm2 each; one reference; and one auxiliary) setup was used to study the addressing possibilities of the method. These electrode microarrays were shown to be able to covalently graft diazonium cations through electrochemical reduction. Cyclic voltammetry and X-ray photoelectron spectroscopy were used to characterize the electrochemical grafting onto our SP graphite surface and suggested that a diazonium monolayer was deposited. Rabbit and human immunoglobulins (IgGs) were then chemically coupled to an aniline derivative (4-carboxymethylaniline), followed by diazotation to form an aryl diazonium function available for the electrodeposition. These modified proteins were both successfully electro-addressed at the surface of the graphite electrodes without cross-talk or interference. The immuno-biochip obtained using this novel approach enabled the specific detection of anti-rabbit IgG antibodies with a detection limit of 50 fmol of protein. A promising strategy to immobilize markedly different biological entities was then presented, providing an excellent spatial specificity of the electro-addressing.